Gastric Cancer Growth Modulated by circSNTB2/miR-6938-5p/G0S2 and PDCD4

Background Gastric cancer (GC) is the third most common cause of cancer-related death worldwide. Increasing studies have indicated that circular RNAs (circRNAs) play critical roles in cancer progression. However, the precise mechanism and functions of most circRNAs are still unknown in gastric cancer. Methods In the present study, we aim to uncover the mechanism by which circRNAs regulate gastric cancer tumorigenesis. By analyzing the microarray data, we screened differential expressed circRNAs in the gastric cancer group and identified a down-regulated circRNA, hsa_circ_0040039 (circSNTB2). Mechanically, circSNTB2 served as a sponge for the miR-6938-5p and up-regulated its expression. Results Meanwhile, G0/G1 switch gene 2 (G0S2) and programmed cell death gene 4 (PDCD4) were identified to be the aim genes of miR-6938-5p, constructing circSNTB2/miR-6938-5p/G0S2 and PDCD4 pathways. Conclusion Taken together, our findings demonstrated that circSNTB2 plays an essential role in gastric cancer by regulating miR-6938-5p through G0S2 and PDCD4 genes. CircSNTB2 could be a promising biomarker for GC diagnosis and targeted therapy.


INTRODUCTION
Gastric cancer (GC) is one of the most common malignant tumors in the clinic and the second cause of cancerrelated mortality, particularly prevalent in east Asia countries [1]. In spite of a steadily declining incidence of GC across the world, the number of new cases continues to increase in China because of population rapid growth and ageing [2,3]. Early detection plays an essential role in GC with a significantly better prognosis compared to advanced GC [4]. However, it is difficult to diagnose because most people typically don't show symptoms in the earlier stages and have poor awareness of risk factors [5]. Besides, less understanding of GC pathogenesis would be another obstacle to treating GC [6]. Prevention and cure of gastric cancer are still challenged in the clinic [7][8][9]. Therefore, searching for new molecular markers to monitor and intervene in gastric cancer carcinogenesis is urgent. Circular RNAs (circRNAs) have been known to play an essential role not only in the ordinary development of organs and tissues [10], but also in the occurrence and progression of human diseases, including cardiovascular diseases [11], neurological dysfunction [12] and cancers [13]. Accumulating evidence has demonstrated that circRNAs are abnormally expressed in various cancer types, such as colorectal cancer [14], hepatocellular carcinoma [15] and breast cancer [16]. What is more, the role of circRNAs in the process of cancer initiation and progression has especially gained concern because circRNAs may cause cancer by interacting with tumor-associated miRNAs, proteins and genes, and by participating in pathophysiological activities. CircRNAs have been recognized as dependable diagnostic and therapeutic molecular biomarkers for cancers [17,18]. Besides, a few researchers have focused on the correlation between circR-NA and GC. Pan et al. have found that ciRS-7/miR-7/PTEN axis plays a significant role in gastric cancer [19]. Has_circ_0000096 and has_circ_0047905 were considered as the bioactive markers of GC due to the area under the ROC curve is 0.82 and 0.85, respectively, [20,21]. More research should be performed to elucidate the link between circRNA and GC.
In this study, we aimed to establish the expression profile of gastric cancer through circRNA microarray chip analysis. Our data indicated that circSNTB2 is involved in gastric cancer through bioinformatics analysis. The miRNA database was further explored to identify circRNA-related dysregulated miR-6938-5p in gastric cancer. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes pathways (KEGG) analysis revealed the potential biology function of miRNA target genes. Finally, a circSNTB2/miR-6938-5p/ G0S2 and PDCD4 regulation network were constructed to selected hub genes and we found that circSNTB2 played a crucial role in the network.

Data Processing
After batch effect normalization, the circRNA and miR-NA microarray data were further analyzed by limma R package to attain differentially expressed circRNAs. P values were adjusted by false discovery rate (FDR). |log 2 (foldchange)| >1 and FDR <0.05 were considered as significant.

Prediction of circRNAs Target miRNAs
Top 10 up and down-regulation circRNA-miRNA interactions were respectively predicted by circBank database (http://www.circbank.cn/index.html). Then we overlapped predicted miRNAs of up and down-regulation circRNAs, respectively. To further improve the credibility of the prediction, we intersected the predicting outcomes with the microarray data by R. And we could screen out the most potential miRNA.

Prediction of miRNAs Target Genes
After screening the miRNA, miRNA verified by expression profiles was considered the most likely potential target miRNA. Then, the screened miRNA was further applied to predict the target genes via four online databases, the Targetscan (http://www.targetscan.org/mamm_31/), miRDB (http://mirdb.org/), Encori (http://starbase.sysu.edu.cn/index. php), miRWalk (http://mirwalk.umm.uni-heidelberg.de/). Target genes of the potential miRNA from the four databases were intersected. To find the most likely potential target genes, the target genes that were predicted by the four databases simultaneously were overlapped with down-regulation genes from GEPIA.

GO and KEGG Pathways of Target Genes
After overlapping results from four databases, target genes were applied to Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes pathways (KEGG) analysis through cluster Profiler R package. Ggplot2 R package was used to visualize the outcomes of the analysis. GO analysis was used for functional analysis of the genes and consisted of biological process (BP), cellular component (CC), and molecular function (MF). KEGG pathway analysis revealed signaling pathway information for the genes.

Survival Analysis and Expression of Target Genes
To obtain Kaplan-Meier survival curves, the most likely potential target genes were analyzed by Kaplan Meier-plotter database (http://kmplot.com/analysis/index.php?p=backgrou nd) [22,23]. The GEPIA database is an online analytical tool based on the TCGA database. The expression of those genes was visualized by GEPIA [24].

Quantitative Real-Time PCR (qRT-PCR)
We used a mortar and pestle to grind the fresh tumor tissue and peritumoral tissue into fine powder under the circumstance of liquid nitrogen [25]. Total RNA was isolated by TRIzol Reagent and quantified by Nanodrop 2000 (Thermo Scientific, Rockford, IL, USA). Single-stranded cDNAs were reverse-transcribed from 1000 ng of total RNA. QRT-PCR was conducted on a CFX96 apparatus (Bio-Rad) with SYBR Green Pro Tap [26]. For each target gene, the relative expression levels of mRNAs were calculated following the 2 −ΔΔCt method and using 18s as a housekeeping gene (primer sequences were listed in Table 1). Repeat at least three times for each sample.

Identification of Differentially Expressed circRNAs
The microarray data of gastric cancer circRNAs was from GSE78092 which used ArrayStar Human Circular RNA microarray V2.0 (GPL21485). After batch effect normalization, we used limma R package to analyze the series matrix file and discovered 199 statistically significant differ-entially expressed circRNAs (DECs) with 53 up-regulated and 146 down-regulated circRNAs (Fig. 1). To enhance the accuracy of prediction, top 10 up-regulated and down-regulated circRNAs were chosen to predict target miRNAs (Tables 2 and 3).

Prediction of circRNAs Target miRNAs and Expression of Target miRNA
The microarray data of gastric cancer miRNAs was from GSE124158 which used 3D-Gene Human miRNA V21_1.0.0 (GPL21263). According to the predicted miRNAs from top 10 of up-regulated and down-regulated circRNAs, we found some overlapped miRNAs, which indicated that these miRNAs have more potential to be affected (Figs. 2  and 3). Among these, hsa-miR-6838-5p has the maximum overlap numbers in the prediction of down-regulated circR-NAs. Therefore, we selected the hsa-miR-6838-5p to be the target miRNA. As the most significantly down-regulated circRNAs, CircSNTB2 had the great potential to interact with hsa-miR-6838-5p. Further, we confirmed that the expression of hsa-miR-6838-5p was higher in gastric cancer group (Figs. 4 and 5).

Screened and Quantified the Target Genes
Based on the outcomes of the GEPIA database, there are 908 down-regulated genes related to GC. Combined the above 700 genes, we identified 15 target genes that have tightly association with hsa-miR-6838-5p (Fig. 8). Then, we found that G0S2 and PDCD4 were downregulated in the tumor tissue by performing qRT-PCR ( Figs. 9 and 10). Thus, G0S2 and PDCD4 were identified as the two potential target genes. Further investigation indicated that the survival rates were dramatically lower in G0S2 and PDCD4 low expression groups compared to the high expression groups with P value less than 0.05 (Fig. 11).

DISCUSSION
Gastric cancer is still the second leading cause of cancerrelated mortality in China because of its highly malignant nature [27]. The understanding of the molecular pathogenesis of gastric cancer has advanced considerably in the last decades. With the recent development of transcriptome sequencing technology, circRNAs have attracted extensive attention from researchers. Mounting pieces of evidence confirmed that circRNAs participate in various bioprocesses in many tumors, such as hepatocellular carcinoma, Breast cancer, non-small-cell lung cancer [28], and bladder cancer [29]. However, few circRNAs have been well mechanistically featured in gastric cancer, and the biological functions of most circRNAs have yet to be elucidated. In our study, we identified a novel circRNA circSNTB2, which is dramatically down-regulated in gastric cancer tissues by analyzing the microarray from gastric cancer tissues and adjacent normal tissues. The under-expression of circSNTB2 in gastric cancer indicates that gastric cancer may be inhibited by upregulating circSNTB2.
CircRNAs have been proven to be the competitive endogenous RNAs (ceRNAs) that could target miRNAs by acting as a miRNA sponge to be involved in human diseases [30]. Here, we predicted that miR-6938-5p as the target of circSNTB2 by analyzing the online database. Furthermore, the expression of miR-6938-5p was found to be elevated in gastric cancer tissue compared with the normal tissue. Gene function analysis, including GO analysis and KEGG pathway analysis was performed for the target mRNAs of the miR-6938-5p. The results of KEGG pathway analysis suggested that PI3K-Akt signaling pathway, MAPK signaling pathway, Human papillomavirus infection, mTOR signaling pathway were significantly enriched which have been already known as the key signals in regulating several cancers including gastric cancer [19,[31][32][33]. Fig. (2). Interaction network about top 10 upregulated circRNAs with target miRNAs. Yellow spots represent circRNAs, pink spots represent miRNAs interacted with 4 circRNAs, blue spots represent miRNAs interacted with 3 circRNAs, green spots represent miRNAs interacted with 2 circRNAs and purple spots represent miRNAs interacted with 1 circRNAs. (A higher resolution / colour version of this figure is available in the electronic copy of the article).

(A) (B)
Further mechanism exploration revealed that G0S2 and PDCD4 act as the target genes of miR-6938-5p. G0S2 was discovered in the early 1990s by Russell and Forsdyke in cultured mononuclear cells which only exist in vertebrates [34]. Many studies have uncovered that G0S2 is a multifaceted protein implicated in cell proliferation, apoptosis, metabolism, and carcinogenesis [35]. Yim has reported that low G0S2 expression in breast cancer, particularly estrogen receptor-positive breast cancer, correlates with increased rates of its recurrence [36]. Besides, G0S2 could function as a tumor suppressor by opposing c-Myc [37]. PDCD4 is another novel tumor suppressor with multi-functions inhibiting tumor cell proliferation, tumor invasion and metastasis [38]. Decreased expression of PDCD4 has been strongly believed to be implicated in the development and progression of many kinds of human tumors, including lung, colon, liver, and breast cancer [39][40][41][42]. Our findings highlighted that the expression of both G0S2 and PDCD4 were inhibited in gastric cancer tissues while compared with the normal tissues. Consistently, the survival rates in the decreased expression of G0S2 and PDCD4 group were significantly lower than G0S2 and PDCD4 high expression group. These results depicted that G0S2 and PDCD4 function as the aim genes of miR-6938-5p playing a central role in the development and prognosis of GC.

CONCLUSION
Taken together, the present data illustrated that circRNAs are aberrantly expressed in GC. Significant down-regulation of hsa_circ_0040039 (circSNTB2) expression in GC tissues was confirmed. Mechanism exploration suggested circSNTB2 can serve as the ceRNA to sponge miR-6938-5p to participate in GC tumorigenesis by modulating the expression of G0S2 and PDCD4. Our research characterized the regulation of circSNTB2/miR-6938-5p/G0S2 and PDCD4 pathways and their role in gastric cancer. In the future, a longitudinal study is needed to perform to identify the potential of circSNTB2 as a biomarker for GC diagnosis and targeted therapy.

AUTHORS' CONTRIBUTIONS
Yongkun Zhou and Qingying Sun designed the study. Baohai Rong wrote the manuscript. Xiqi Chen, Guangdong Xie and Letian Han collected all data. Hanhan Chen and Baohai Rong analyzed the collected data.

ETHICS APPROVAL AND CONSENT TO PARTICI-PATE
Not applicable.

HUMAN AND ANIMAL RIGHTS
No animals/humans were used for studies that are the basis of this research.

CONSENT FOR PUBLICATION
Not applicable.

AVAILABILITY OF DATA AND MATERIALS
Tha authors confirm that the data and supportive information are available within the article.

FUNDING
None.